The TRP superfamily comprises a large group of cation channels with homology to the Drosophila TRP channel, which plays a key role in phototransduction. Among the seven TRP subfamilies, the TRPP proteins are of particular interest because mutations in the founding member, PKD2, result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). TRPP channels are evolutionary conserved and in other organisms and play a key role in male fertility. A number of recent studies have demonstrated that members of the TRPP family can be found in ciliated structures where they are thought to participate in mechanosensory signal transduction pathways. Despite intensive investigation, however, there are numerous aspects of TRPP biology that remain controversial including the subcellular localization of TRPPs and the identity of proteins that participate in TRPP signaling pathways. Many attempts have been made to address these questions using heterologous expression systems. However, the results have not always been consistent and they have been difficult to verify in vivo. We have developed a Drosophila model system with a targeted mutation in the fly homologue of PKD2 (named amo for Almost There). Male flies carrying a null mutation in amo were found to have normal amounts of motile sperm but are infertile due to an inability of sperm to be stored in the female storage organs. In addition we find that Amo is concentrated at the tip of the sperm flagella suggesting a conserved role for TRPPs in axonemal structures. This Drosophila system offers us the opportunity to use a genetically tractable model organism to correlate in vitro findings with in vivo analyses. In this proposal we will use a multidisciplinary approach to address three unresolved issues in TRPP biology: 1) the developmental regulation of TRPP2 localization in vivo 2) The requirement of cation channel activity for TRPP2 function in vivo and 3) the identification of upstream and downstream components of the TRPP signaling cascade. We anticipate that these studies will provide new information about the critical functional and signaling properties of the TRPP class of ion channels. [unreadable] [unreadable] [unreadable]